Aphanothece sp. as promising biostimulant to alleviate heavy metals stress in Solanum lycopersicum L. by enhancing physiological, biochemical, and metabolic responses

Heavy metals (H.M) are a major environmental concern around the world. They have harmful impact on plant productivity and pose a serious risk to humans and animals health. In the present study, we investigated the effect of Aphanothece crude extract (ACE) on physiological, biochemical, and metabolic responses of tomato plant exposed to 2 mM Pb and Cd. The results showed a significant reduction of tomato plant weights and perturbation in nutrients absorption under 2 mM Pb and Cd conditions. Moreover, ACE treatment showed a significant enhancement of plant biomass compared to plants under Pb and Cd. On the other hand, ACE application favoured H.M accumulation in root and inhibited their translocation to shoot. In addition, ACE treatment significantly enhanced several stress responses in plant under Pb and Cd stress such as scavenging enzymes and molecules: POD, CAT, SOD, proline, and polyphenols etc. Furthermore, ACE treatment showed remodulation of metabolic pathways related to plant tolerance such as wax construction mechanism, particularly SFA, UFA, VLFA, alkanes, alkenes, and sterols biosynthesis to enhance tolerance and resistance to H.M stress. In the present study, we emphasized that ACE alleviates H.M stress by minimizing metal translocation to above-part of plant and enhancing plant growth, nutrients absorption, and biochemical responses.


Effect of microalgae ACE extract on tomato growth under Pb and Cd stress. The effect of ACE
on tomato plant growth under heavy metals stress was investigated in the present study, ACE application at sowing was not able to improve seed germination (data not provided here). While plants treated at four leaves stage has a remarkable difference between treatments. Thus, we used to apply our ACE on vegetative stage. In results, tomato plants exposed to 2 mM Pb and Cd stress showed no significant difference in tomato dry weights even for roots or shoots under 2 mM Pb. However, a significant reduction was noted in the dry weight of shoots and roots under 2 mM Cd by 29.25% and 39.48%, respectively compared to control (Fig. 1). Furthermore, the treatment of tomato under 2 mM Cd by ACE showed a significant recovery of dry weights shoot and root by 26.11% and 40.35%, respectively compared to plants under 2 mM Cd. by GraphPad prism 9 software, while "*" indicates statistical significance (P < 0.05); "**" indicates statistical significance (P < 0.01); "****" indicates statistical significance (P = 0). Effect of ACE on plant antioxidant system against Pb and Cd stress. In the present study, we investigated the effect of ACE on plant antioxidative system under 2 mM Pb and Cd. Tomato leaves showed significant elevation of H 2 O 2 level with 63.45% and 110.15% under 2 mM Pb and Cd stress, respectively. Moreover, 2 mM Pb and Cd showed an increase of Malondialdehyde (MDA) level with 98.67% and 66.24%, respectively (Fig. 3a,b). Moreover, 1% ACE supplementation reduced significantly H 2 O 2 and MDA level by 15.41% and 64.79% for Pb and 56.34% and 45.11% for Cd, respectively. Furthermore, the exposure to 2 mM Pb and Cd showed an accumulation of proline and polyphenols by 43.01% and 8.20% under 2 mM Pb, respectively and 58.21% and 9.26% under 2 mM Cd, respectively (Fig. 3c,d). In addition, ACE supplementation showed reduction in proline and polyphenols accumulation under stress by 31.46% and 7.56% under Pb + ACE in comparison to 2 mM Pb. For Cd, ACE application showed reduction of 35.98% and 15.16% in proline and polyphenols, respectively. 2 mM Pb and Cd exhibited significant increase of soluble sugar and proteins content in tomato leaves (Fig. 2e,f)  by GraphPad prism 9 software, while "*" indicates statistical significance (P < 0.05); "**" indicates statistical significance (P < 0.01); "***" indicates statistical significance (P < 0.001); P "****" indicates statistical significance (P = 0)].  [Figure generated by GraphPad prism 9 software, while, "*" indicates statistical significance (P < 0.05); "**" indicates statistical significance (P < 0.01); "***" indicates statistical significance (P < 0.001); "****" indicates statistical significance (P = 0)].   5). Our results showed translocation inhibition from root to shoot, which tomato plants exposed to 2 mM Pb showed a Translocation Factor (TF) exceed 1, which was 3.01 ± 0.037. Moreover, ACE application to plant stressed by 2 mM Pb showed reduction in TF, which was 1.11 ± 0.028. For Cd, ACE showed in inhibition of Cd uptake and accumulation and both Bioconcentration Factor (BCF) and TF doesn't exceed 1 ( Table 3).

Discussion
In the present study, the effect of ACE on physiological, biochemical, and metabolic responses under heavy metals stress has been investigated. ACE application showed a significant recovery of shoot and root dry weights by 26.11% and 40.35%, respectively after reduction under 2 mM Cd by 29.25% and 39.48%, respectively compared to control. These results are in agreements with the finding of Abd El-All et al. 23 showed that the treatment by seaweed extract (SWE) had a positive role in recovery to deleterious effect of heavy metals on tomato plant growth in comparison with control. respectively. In the light of this point, microalgae based biostimulant showed positive effect on plant growth and yield by reducing stress and restoring previous damages 24 . The biostimulant effect of microalgae extract was reported in numerous studies. Li et al. 11 , showed that 1% of microalgae extract enhanced significantly bean seedling growth. Tomato plants treatment by microalgae and cyanobacteria crude extract (CBEs) showed significant growth enhancement, particularly Aphanothece sp. which improved significantly root and shoot DW (34.81% and 58.69%), respectively 20 . The stimulation effect of microalgae extract may be due to the presence of plant growth promoting substances such as: macro-and micro nutrients, amino acids, fatty acids, polysaccharides, phytohormones etc., which can affect cellular physiology of plants enhancing plant growth and productivity 11,[24][25][26] .
Furthermore, ACE supplementation showed reduction in proline and polyphenols accumulation under stress by 31.46% and 7.56% under Pb + ACE in comparison to 2 mM Pb. For Cd, ACE application showed reduction of 35.98% and 15.16% in proline and polyphenols, respectively (Fig. 3c,d). In line of our results, selenium application as biostimulant at 1 µM under Cd stress reduced proline accumulation in tomato leaves 8 .
Scavenging enzymes are another barrier of antioxidative system played to reduce free radicals 31 . In the present study, we noted that ACE supplementation showed neutralization of scavenging enzymes (SOD, CAT and POD) after their elevation under H.M stress (Fig. 3e-g). In accordance, seaweed extract (SWE) application by foliar spray mode reduced significantly POD activity and in result recovered the deleterious effect of H.M 23 . Microalgae biostimulant contains bioactive molecules such as proline, betaines, phytohormones which can regulate plant redox homeostasis leading to stress resistance and tolerance 26  Our results showed that ACE can improve plant tolerance to H.M stress by stimulating plant oxidative system at early stage. ACE treatment alone showed elevation of H 2 O 2 by 68.37% (Fig. 3a). However, ACE supplementation with Pb or Cd stress showed reduction of H 2 O 2 , MDA and scavenging enzymes activities after their elevation under Pb and Cd stress. Which indicate that plant early immunized for any stress by the first elevation of H 2 O 2 from ACE. Furthermore, microalgae biostimulant act as elicitor contain bioactive molecules such as proline, betaines, phytohormones which can regulate plant redox homeostasis molecules leading to stress resistance and tolerance 33,34 .
ACE supplementation to tomato plants under Pb and Cd showed increase of SFA and MUFA, while decrease PUFA, alkanes and sterols after their augmentation under Pb and Cd stress (Fig. 4, Tables 1 and 2). Moreover, ACE application alone showed increase of PUFA, alkanes and sterols, which have a protective role against stress. PUFA, alkanes and sterols are the major metabolites that have a role in plant tolerance to abiotic and biotic stress 35 . PUFAs are the main compound of membrane, which keep plasma membrane permeability, integrity, and fluidity. They have also a role in ROS generation via activation of Nicotinamide adenine dinucleotide phosphate NADPH oxidase 36,37 . PUFA act also as the precursors of Lipoxygenases (LOX) pathway resulting in oxylipins biosynthesis after their peroxidation 38 . Oxylipin play the role of signaling and defense molecules 39 . In addition, the oxylipin induce expression of genes involved in the biosynthesis and accumulation of secondary metabolites 40,41 . Moreover, Alkanes are also a compound of membrane and wax cuticle, which play a vital role as a defence barrier against abiotic and biotic attacks 42,43 . Sterols have a role in membrane formation and preservation, against abiotic and biotic shocks 44 . Plants sterols can act as precursors of Brassinosteroids (BRs) 45 . Moreover, BRs improve plant tolerance to metal, thereby increasing crop yield and quality 46 . They can eliminate toxic metal via assisted phytoremediation system by plant growth regulators. BRs reduce H.M uptake via membrane permeability alteration, improve soluble proteins and increasing ATPase activity 47 . Moreover, they help in metal detoxification by enhancement of antioxidative system via scavenging enzymes and proline accumulation 46 .The accumulation of these metabolites can explain the role of ACE to alleviate H.M stress regarding to metabolites accumulated in comparison with plants under metal stress alone. In accordance, Rachidi et al. 19 reported that microalgae polysaccharides showed redistribution of metabolites with improvement of lipids, alkanes, and sterols in tomato plants. Moreover, the treatment of tomato plant by liquid microalgae extract showed enhancement of SFA especially palmitic and stearic acid which are the first stage of de novo lipid synthesis 20 .
To investigate the effect of ACE on Pb and Cd accumulation, tomato plants exposed to 2 mM of Pb and Cd alone and combined with ACE. Thus, ACE supplementation under Pb significantly enhanced the accumulation of Pb in root by 88.78% compared to 2 mM Pb. Moreover, in shoot ACE significantly reduced the accumulation of Pb by 30.18% compared to 2 mM Pb. In accordance, spent mushroom compost (SMC) used as biostimulant of Megathyrsus maximus in contaminated soil improved significantly H.M uptake and remediation including Pb and Cd 48 . Furthermore, ACE significantly reduced Cd concentration in root and shoot by 75.77% and 44.69% compared to 2 mM Cd. In line of our results, the treatment by Ca (10 mmol L −1 ), salicylic acid (100 µmol L −1 ) and epi-brassinolide (1 mol L −1 ) in combination showed reduction of Cd concentration in root, stem and leaf 32 .
ACE application showed reduction of metal accumulation in root or/and shoot, which make ACE to be used for phytostabilisation strategy Indeed, the use of biostimulant directly in contaminated soil may have metal chelator effect, which enhance metal solubility and uptake by plants 48 . It may be due also to nutrients competition www.nature.com/scientificreports/ which ACE enhance nutrients uptake. In this regard, Gharaibeh et al. 49 showed that zinc (Zn) combination with Cd may reduce metal concentration in different part of tomato plants. Moreover, potassium (K) supplementation at 310 ppm significantly reduced Cd translocation from root to shoot in tomato 50 . K supplementation confers plant exposed to Cd a positive response 32 . K can efficiently reduce Cd-toxicity and improve health of plant by enhancing photosynthesis activity and the biosynthesis of photosynthetic pigments 50 . Calcium (Ca) also reported as competitor ion of metals. The application of Ca at 2.5 mM in combination with Pb at 2.5 mM in soybean significantly reduced Pb accumulation in roots 51 .
Our results showed translocation inhibition from root to shoot, which tomato plants exposed to 2 mM Pb showed a TF exceed 1. Moreover, ACE application to plant stressed by 2 mM Pb showed reduction in TF. For Cd, ACE showed in inhibition of Cd uptake and accumulation and both BCF and TF doesn't exceed 1 ( Table 3). Even that the concentration found in tomato plants parts doesn't exceed the limit imposed by the FAO/WHO for both Pb and Cd 52 . However, tomato showed the aptitude of metal uptake and translocation to the above part of plant, which represent a health risk to animal and humans. For this reason, an inhibition of metal uptake and translocation to the above part is necessary. Indeed, our study showed the potential of ACE as biostimulant of plant tolerance to heavy metals, which can minimize Pb and Cd uptake and translocation to the above part. In accordance, Gharaibeh et al. 49 reported that fruits showed low Cd accumulation than shoot and root. Moreover, Eid et al. 53  In this regard, we investigate the effect of ACE on nutrients uptake and redistribution under Pb and Cd stress (Figs. 6 and 7). ACE supplementation under 2 mM Pb and Cd increased NPK concentration in root and shoot compared to control. NPK are the most important nutrient for plant growth and development 54 . N is an important mineral element for plant productivity, which found mostly in nitrate, ammonium and organic molecules such as amino acids 55 . In the present study metal stress induced increase in N content in root and shoot even when added ACE. Moreover, our results showed increase of soluble proteins and enzymes activity which indicate the functional use of N assimilated by plant under each treatment. In accordance, Schreiber et al. 56 showed the potential of Chlorella vulgaris as physiostimulators to enhance N and P accumulation in wheat plant. Generally, K deficiency decreased Chl a and b biosynthesis, which negatively affect plant growth and development 54 . In the present study, metals stress induced increase in K level in plant. However, we found a decrease in Chl a and b content in plant. This finding indicates that Pb and Cd affect the function of nutrients in photosynthetic pigments not the essential nutrients uptake. In accordance, Li et al. 57 reported significant increase of K concentrations in welsh onion under 2.5 mg kg −1 Cd. Contrary, H.M stress showed a significant reduction of NPK concentration in soil irrigated with wastewater respectively 58 . Furthermore, the increase of some mineral concentrations under H.M stress might be attributed to their incorporation for H.M detoxification 57 . In conclusion, ACE may enhance nutrients uptake and translocation to minimize the uptake of Pb and Cd and detoxify these metals when they are present in plants parts, in turn enhance plant tolerance to metal stress.
The effect of metal and treatment by ACE on other nutrients concentration was shown in Fig. 7. ACE supplementation significantly increased the concentration of Zn (92.31%), Mg (31.78%), Cu (34.39%) and Cr (42.85%) after their reduction a under 2 mM Pb. Furthermore, ACE supplementation significantly enhanced concentration of Ca and Cu by 644.29% and 26.39%, respectively after their reduction under 2 mM Cd. In this regard, Asemoloye et al. 48 reported that the use of SMC at 20% as biostimulant of Megathyrsus maximus enhanced nutrients status in contaminated soil by H.M. The use of C. reinhardtii and C. sorokiniana as biostimulant improved Mn and Cu, concentration in maize seedling 59,60 . Kusvuran 60 showed that plant leaves treatment by C. vulgaris extract may affect positively plant nutrients content. The use of biostimulants may enhance nutrients uptake, which improve plant growth and productivity. Moreover, limiting the use of chemical fertilizers and protecting environment 21 . Numerous studies showed that biostimulant may protect plants from the excess and deficiency of nutrients 21,60,61 . Vernieri et al. 62 reported that the application of biostimulant reduced the nitrate content in leaves. Moreover, microalgae may act as physioactivators, which can stimulate nitrate reductase and other enzymes incorporated in minerals absorption and transformation in plants 60 . Microalgae have bioactive molecules which has a major role in agriculture such improving nutrient uptake, physiological status, crop performance and abiotic stress tolerance 63,64 .

Conclusion
Pb and Cd treatment reduced tomato growth and photosynthetic pigments through the generation of ROS and nutrients uptake disruption. However, ACE application showed corrective effect which increased tomato plant growth under Pb and Cd stress and enhance photosynthetic pigments. Tomato exposure to Pb and Cd showed increase in H 2 O 2 , which in results increase lipids peroxidation (MDA). ACE treatment showed reduction of H 2 O 2 , MDA and scavenging enzymes activities after their elevation under Pb and Cd stress. 2 mM Pb and Cd stress showed the reduction of SFA and MUFA, while increased PUFA, alkanes and sterols compared. However, ACE treatment under Pb and Cd stress showed increase of SFA and MUFA, while decrease PUFA, alkanes and sterols. Tomato plant showed significant accumulation of Pb and Cd in root. As well as a significant accumulation in shoot. Whereas ACE application favoured heavy metals accumulation in root and inhibited their translocation to shoot.Thus, ACE can be used as biostimulant of plant tolerance to heavy metals by minimizing H.M translocation to the aboveground part and can be used also in phytostabilisation strategy. Pb and Cd inhibited nutrients uptake and their distribution in plants. ACE application showed a positive effect in nutrients uptake and translocation under Pb and Cd stress. ACE alleviate metal stress by enhancing antioxidative system and nutrients status from the uptake to redistribution in different plant parts.  Table S3 by  Biochemical parameters analysis. Photosynthetic pigments extracted and determined according to Xiong 69 method. 0.1 g of fresh tomato leaves were grinded in liquid nitrogen using a mortar and pestle. The concentration of pigments was calculated according to the following formulae 70 : Soluble proteins in tomato leaves was extracted according to Fleurence 71 and determined following Bradford 66 method by recording absorbance at 595 nm by Spectra Max Plus Molecular Devices spectrophotometer using BSA as standard. Total sugar was determined according to phenol-sulfuric method 67 . Total phenols were calorimetrically determined using Folin-Ciocalteu reagent as described by 72 . www.nature.com/scientificreports/ (PVP) and 0.5% (v/v) triton X-100. The crude protein concentration in supernatant was estimated by Bradford method using BSA as standard for protein quantification 66 . SOD activity was determined according to Beauchamp and Fridovich 76 method. CAT activity was determined according to Aebi 77 method. Then, POD activity was measured following guaiacol oxidation method 78 .

GC-MS metabolomic analysis.
To explore the tomato plants metabolomic responses under heavy metals (Pb and Cd) stress and ACE treatment, GC-MS method was performed following the described in 19 . Metabolomic analysis carry out by gas chromatography (GC) (Agilent 7890 A Series GC) coupled to mass spectrometry (MS) (Agilent 5975C) equipped with multimode injector and HP-5MS column with dimension of 30 m 250 mm 0.25 mm and electron impact ionization.
Heavy metals and mineral nutrients analysis in tomato tissues. 0.4 g of each ground sample in triplicates was placed in individual tubes and digested with 2.5 mL of H 2 SO 4 at 100 °C for 2H, followed by adding 2 mL of 30% hydrogen peroxide (H 2 O 2 ) carefully to each tube to complete digestion at 330 °C for 2H. Finally, after the sample solutions were cooled down, the volume was adjusted to 75 mL, and then filtered. The concentrations of NPK were determined by flow ionic analysis, Sakalar scan++ system at the Algal Biotechnology Center, Mascir, Morocco. 0.4 g of dry root and shoot were put it into digestion tube placed on aluminium heating block digester in triplicate. The digestion and mineralization of samples was done according to Gupta 79 method. Heavy metals and mineral nutrient concentrations were estimated using inductively coupled plasmaoptical emission spectroscopy (ICP-OES); (iCAP-7000 Duo, Thermo Fisher Scientific) at the Cereal and Legume Quality Laboratory, ICARDA, Morocco. Bioconcentration factor (BCF) and translocation factor (TF) were used to evaluate the potential of tomato to accumulate H.M within their root and the translocation to the above part. BCF and TF were determined following equation publishes in 80 : Statistical analysis. Statistical analyses were performed using SPSS (IBM SPSS statistics 22). Descriptive statistics and significant differences of the mean values were determined using one-way ANOVA with a posthoc Tukey's and Duncan's tests at a significant level of 0.05. While "*" indicates statistical significance (P < 0.05); "**" indicates statistical significance (P < 0.01); "***" indicates statistical significance (P < 0.001); "****" indicates statistical significance (P = 0). Graphs were generated by GraphPad prism 9 software (https:// www. graph pad. com). All experiments in the work were performed in three replicates; the results were represented as arithmetic mean ± standard deviation.

Data availability
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